Fragmentation of Ceruloplasmin Following Non-Enzymatic Glycation Reaction

Abstract

Bovine ceruloplasmin underwent fragmentation following non-enzymatic glycosylation. Western blot and ELISA analyses indicated that a polyclonal rabbit antiserum to hexitollysine reacted with bovine ceruloplasmin after incubation with 0.1 M glucose. The same fragmentation was seen upon exposure of the protein to a hydrogen peroxide bolus. Both catalase and EDTA blocked peroxide-dependent fragmentation. Incubation with glucose resulted in a time-dependent release of Cu²⁺. The released Cu²⁺ appeared to participate in a Fenton-type reaction to produce hydroxyl radicals, which effected the fragmentation. Hydroxyl radical scavengers such as thiourea, mannitol, methionine, and formate inhibited this cleavage. ESR spectral studies also supported participation of hydroxyl radicals. Inhibition by EDTA of the fragmentation induced by an H₂O₂bolus also supports a role for copper in a Fenton-type reaction. Taken together these results suggest that reactive oxygen species, such as superoxide anion and H₂O₂, were formed by the Maillard reaction which led to hydroxyl radicals being produced by a copper-dependent Fenton-type reaction. Both processes are likely to be involved in the fragmentation of ceruloplasmin.

ceruloplasmin, copper, glycation, hydroxyl radicals